The emergence of novel strains of influenza A virus is an ever-present threat to human health worldwide. Currently, the cornerstone for the prevention of serious disease is intramuscular vaccination with inactivated influenza strains. The effectiveness of the vaccine is due to the induction of virus-specific serum antibodies (Abs), and also the generation of memory B cells (MBCs) that respond to "recall" antigen with rapid and vigorous Ab production. Despite the key role of MBCs in resistance to infection, quantitative aspects of MBC generation have received little attention. The major objective of the current proposal is a quantitative analysis of the MBC pool generated by influenza A virus infection of the respiratory tract, or by vaccination with inactivated virus. Studies will be undertaken using a mouse model system, and a limiting dilution assay that has been adapted for the determination of influenza-specific MBC frequencies. Aim 1 will focus on the frequency, anatomical distribution, and Ab isotype expression of MBCs induced by live virus infection. The hypothesis that mucosal infection generates a concentration of MBCs at mucosal surfaces will be tested. This work will be extended under Aim 2 to a characterization of the MBC pool induced by vaccination. The relationship between route of vaccine administration and MBC distribution and isotype expression will be investigated, and the effect of boosting regimens in "optimizing" the MBC pool will be examined. Systems established in Aims 1 and 2 will provide the basis for achieving Aim 3, in which the question of whether a heterologous virus infection can perturb a pre-existing, influenza-specific MBC pool will be addressed. The studies in this proposal will enhance our understanding of immunity to all viruses that target the respiratory tract, and will provide a basis for rational vaccine development. In addition, the systems established could be used in many other ways to address mechanistic questions related to the generation and maintenance of B cell memory.